Production of hybrid cottonseed by chemically inducing male-sterility



United States Patent PRODUCTION OF HYBRID COTTONSEED BY CHEMICALLYINDUCING MALE-STERILITY Frank M. Eaton, Riverside, Calif., and DougalHarold McRae, Levittown, Pa., assignors to Rohm & Haas Company,Philadelphia, Pa., a corporation of Delaware No Drawing. ApplicationAugust 19, 1958 Serial No. 755,898

7 Claims. (Cl. 47-5) This invention relates to the production of hybridcottonseed. It is based on the discovery that certain chlorinatedaliphatic acids and their water-soluble salts adversely affect theproduction of fertile pollen in the cotton flower without apparent,detrimental effect upon the ovules.

It has been known that significant improvement in plant characteristicsand yield of cotton can be obtained from cottonseed produced underconditions conducive to a high percentage of cross-pollination. Cottongeneticists have also long realized that, if male-sterility could beinduced or imparted to cotton plants, improved hybrid cottonseed wouldbe obtainable which would greatly improve the quality and quantity ofcotton from the first generation plants. Heretofore, however, no meanswere available for inducing or producing male-sterility except bylaborious hand-emasculation of the cotton flower.

It is also known that the chlorinated aliphatic acids and their salts ofthe kind found useful in the practice of this invention affect in oneway or another the growth characteristics of many plants. Some of themhave been proposed for use as herbicides or to otherwise controlundesirable plant growth. They have not, however, heretofore been knownto have selective action on the development or functioning of the maleor female gametes.

The present invention is based upon the discovery that, when used oncotton in the manner hereinafter described, these chlorinated aliphaticacids and salts so affect the flowering of the plant that there areproduced flowers otherwise normal but containing little or no pollen orcontaining a sterile or substantially sterile pollen. By so inducingmale-sterility while preserving fertile ovules, plant specimens areobtained admirably suited to crosspollination by natural means.

The compounds which may be used in the practice of this invention arethose compounds which in watersolution form anions of a chlorinatedaliphatic acid which contains from two to four carbon atoms and at leasttwo chlorine atoms. Specifically, these acids are 2,3-dichloroisobutyn'cacid, trichloroacrylic acid, 2,3-dichloropropionic acid,2,2,3-trichloropropionic acid, trichloroacetic acid,2,2-dichloropropionic acid, 2,2,3-trichloro-n-butyric acid, anddichlorosuccinic acid. These acids may be used as the free acid or inthe'form of their salts, examples of which are the sodium, potassium,iron, aluminum, cadmium, calcium, nickel, mercury, copper, zinc,manganese, and barium salts, and the ammonium salts formed with ammonia,amines or quaternary ammonium groups, including monomethylamine,dimethylamine, trimethylamine, the ethylamines, ethanolamine,dimethylethanolamine, morpholine, pyrrolidine, or pyridine, or suchquaternary groups as tetramethylammonium, benzyltrirnethylammonium,dibenzyldimethylammonium, methylpyridinium, or methylmorpholinium,didodecenyldimethylammonium, N-dodecyl-N- benzyl-N,N-dimethylammonium,N-dodecylbenzyl-N,N,N- trimethylammonium, oroctylphenoxyethylbenzyldimethylammonium.

In practicing the invention, the. area. in which the bybrid seed is tobe produced is planted with at least two varieties of cotton plant. Thevarieties may be of the same species or may be varieties of diiferentspecies. Thus, two varieties of American-Egyptian may be crossed or avariety of American-Upland may be crossed with an Egyptian cotton. SeaIsland and Upland crosses appear to be of particular interest. The termvariety as hereinafter used should be understood as meaning subspeciesof a species and also, if there be a species without recognizedsubspecies, then the species itself as the sole variety of the species.The two or more varieties are planted in such manner that all plants ofeach variety are in close proximity with each other, preferably inalternate rows or in an arrangement of two rows of the varietyto bevrendered male-sterile alternating with one row of the second variety.Such arrangements allow for ready access to each variety withpower-driven equipment but this invention does not depend on theparticular arrangement and any other systematic mixed planting of thearea may be practiced which on the one hand places the two varieties.

sufficiently close to each other to encourage cross-pollination yetsufliciently separated to allow for the spraying or dusting of onevariety without harmful contamination of the other.

To effect male-sterility or substantial male-sterility in.

parently identical treatment. The optimum dosage will also vary with theparticular compound being used as-thcy are not all equally active andsome are more phytotoxic to the cotton plant than others. A goodpractical rule is to apply the chlorinated aliphatic acid or salt at arate at which slight injury to the plant occurs from which it willrecover, or at a rate slightly below the point of injury.

In the case of 2,3-dichloroisobutyric acid our experience has been thatinjury to the cotton plant begins to show at an application rate ofabout 8 pounds per acre. At

double this rate the injury is significant, but the. plants. recover. Asimple test on one or two plants will readily show whether theparticular spray concentration and ap plication rate cause severeinjury. In the case of species more susceptible to injury or whenapplication is made under circumstances conducive to plant injury, thedosage may be applied in two or more sprayings or dustings with only aportion of the total dosage in each ap-' plication whereby theconcentration of active principle in the plant system is built up instages or is maintained.

at an effective level. Useful results are obtainable at considerablylower rates and, particularly when repeat.

spraying is practical, as little as one pound peracre will producechemical-emasculation. I

The chlorinated aliphatic acids or salts thereof should be applied as awater-solution or as dust.. The concentration of active ingredients inthe water'is less important] than the rate of application of activeingredients per acre of treated area and may vary over a wide range from0.1% to 5%, with the lower concentrations being particularly useful whentwo or more applications are made. We prefer a concentration of from0.5% to 2%. Wetting agents such as dodecylbenzene sodium sulfonate,sodium. dodecyl sulfate, dodecyltrimethylammonium chloride, or anoctylphenoxypolyethoxyethanol maybe added if de sired but such agentshave a tendency to increase the phytotoxicity of the spray. -For thepreparation of suitable dusts, the active ingredient is combined withinert carriers such as finely particled clays, talc, pyrophyllite,diatomaceous earth, and magnesium carbonate with or without the additionof dispersing or Wetting agents.

The timing of the application of the chlorinated aliphatic acids andtheir salts to the cotton plant does not appear to be critical. Thesechemicals are absorbed by and they or their derivatives are translocatedwithin the plant and their efiectiveness remains for several weeks afterapplication. It has been observed that treating the soil before or afterplanting results in the chlorinated aliphatic acids and their saltsbeing absorbed by the plant and producing physiological responses.Preferably, the chemicals are applied after the plants have becomeestablished and before the first flower buds have opened. The followingexamples illustrate the practice of the invention.

Example 1 Greenhouse Empire cotton plants were sprayed once with a 1.2%solution of the sodium salt of 2,3-dichloroisobntyric acid and thegrowth of the plants observed over a five-week period. During this timethe plants uadrupled in size and many new branches were produced thatbore flowers. The flowers appeared normal except that they produced noviable pollen. Some of the flowers on the treated plants werehand-pollinated with pollen from plants that had not been treated withchlorinated aliphatic acid. The flowers thus pollinated produced normalbolls with viable seed. Those flowers that were not hand-pollinatedproduced young parthenocarpic bolls, which the plant shed.

Example 2 An experimental plot was planted with alternate rows ofred-leaf and green-leaf cotton. These were selected for the test becauseit is known that, when flowers of green-leaf varieties are fertilizedwith red-leaf pollen, the resulting seeds produce seedlings having redhypoeotyls and red cotyledons when exposed to bright light. Fourvarietes of green-leaf cotton were selected and each was planted in arow forty feet long between adjacent rows of red-leaf cotton. There wereten-foot row segments of red-leaf cotton on the ends of each row ofgreen-leaf variety. When the plants were to inches tall, about one weekin advance of the first flowers, the four green-leaf varieties weresprayed with a 1% aqueous solution of the sodium salt of 2,3-dichloroisobutyric acid at a rate which used one ounce of dry chemical per100 feet of row. This is equivalent to an application rate ofapproximately eight pounds per acre. (In considering the dosage rate,only those plants actually sprayed are taken into account. Thus, sincein this test only alternate rows were sprayed, an application rate ofeight pounds per acre requires only four pounds of chemical per acre ofplanted area.) This dosage gave little evidence of plant injury and, asit rained after the first spraying, a second application of /2% solutionusing the same carrier volume was sprayed on the same plants thefollowing day. Starting about two weeks after the first bolls hadopened, the greenleaf cottons were picked three times at bi-weeklyintervals. The seeds from thesepickings were planted in greenhouse traysand the following percentages of seedlings having red hypocotyls wereobtained:

These data indicate that, except for the variety Acala 4-42, thetreatment produced a high degree of malesterility in the green-leafcottons. The Acala 4-42, however, produced more greenthan red-leafseedlings, which indicates that this variety had not been renderedmale-sterile. With the pollen from the green-leaf Acala available in thetest planting, it would not be expected that the seedlings from theother varieties would all be red, but even with this consideration thetests establish a high degree of hybridization. Subsequent tests performed in the greenhouse have established that, by using a somewhat morephytotoxic treatment on the Acala variety, male-sterility is induced.

The fact that the Acala variety possesses a higher threshold value forinduced male-sterility has the important advantage that this variety maybe crossed with a variety having a lower threshold value by amodification of the process herein described wherein the varieties ofdifferent threshold value are planted in close proximity to each otherand the entire field sprayed or dusted at a dosage level sufficientlyhigh to induce male sterility in the variety of lower threshold valuebut below the level at which sterility would be induced in the Acalavariety. In this modification it is unnecessary to avoid or minimizecontact of the spray or dust with the variety intended as male parent.

Test data have also established that the physiological responses ofcotton to the sodium salt of 2,3-dichloroisobutyric acid arecharacteristic of the responses induced by the free acid and by othersalts and by the other chlorinated aliphatic acids and their saltsheretofore mentioned. While some variation is to be expected among thedifierent specific chemicals, even as such variation exists among thedifferent varieties of cotton, these variables or their practical efiectcan be reduced considerably, if not eliminated, by following one of therules heretofore mentioned of determining the dosage which will causesevere injury on a few plants in the area to be treated and applying adosage sufiiciently below this level to be safe either by reducing thetotal amount of chemical applied or by applying it in two or moresprayings of safe dosages each. When repeat applications are made, caremust be observed to prevent a build-up of the chemical in the plantsystem to the point where serious toxic effects are produced. It is alsowithin the purview of this invention to use different specificchlorinated aliphatic acids or their salts in the different sprays whena plurality of sprays are to be applied as is also the use of a mixtureof these chemicals in a single spray.

Since by the proper use of the chlorinated aliphatic acids and theirsalts male-sterility can be induced in the cotton plant, it is nowpossible to hybridize two varieties or species of cotton by treating theone type in the manner herein described and cross-pollinating from theuntreated second type. The cross-pollination may be accomplished by handbut preferably by natural means through pollentransporting insects, andfor this purpose a good precaution is to have in the area a good supplyof bees. It will be apparent from the foregoing that this invention isnot in a particular cross-breeding of cotton strands but rather in aprocess of general application appropriate for the production of hybridcottonseed from many difierent crosses. This invention provides theindustry with a means of producing commercial quantities of a widevariety of hybrid cottonseed. It may be expected that some crosses willpossess superior qualities from the veiwpoint of fiber quality orquantity and others from the viewpoint of the quality or quantity of theoil obtainable and some crosses will undoubtedly produce inferiorhybrids.

Example 3 spray per acre1.6 pounds per acre of this salt. The varietieshere used are Stoneville, Empire, Pope, and Cal 51. In the same testareas there are cotton plants of a particular genetic constitution toprovide pollen with a red marker gene (Acala).

When the flowers open, evident stamen abnormalities result in a lack ofviable pollen in each of the flowers then opening. Nevertheless, theflowers set normal bolls as a result of cross-fertilization with pollenfrom adjacent red marker cotton.

After 28 days observations are made to determine the percentage ofcross-fertilized bolls.

From these observations it is found that 67% to 91% of the bolls arehybrid. Germination tests of the seeds contained in these bollsincontrovertibly demonstrate their hybrid nature through the presence ofthe red leaf seedling characteristic.

(b) In another test a 0.5% solution of sodium trichloroacrylate issprayed on Acala 442 cotton plants at 80 gallons per acre-3.2 pounds ofthis compound per acre. The time of spraying is about days prior to theopening of the first flower. When flowers open, stamen abnormalities areobserved accompanied by a lack of viable pollen. In general otherwise,the flowers appear normal. Cross-fertilization is effected by bees usingpollen collected from adjacent untreated red marker variety. Thefertilized bolls develop and the hybrid nature of the seed therein isverified by subsequent germination tests in which red leaf seedlings areinvariably produced.

Example 4 (a) Plants of the variety identified as Pirna S1 are grown inthe vicinity of the above-noted red marker cotton plants. About 14 daysbefore expected blossoming the Pima S1 plants are sprayed with anaqueous 1% solution of 2,3-dichloropropionic acid. Instead of the freeacid some plants are sprayed with a 1% solution of the sodium salt ofthis acid. About 20 days after spraying flowers are produced on theplant with aborted anthers, and 28 days thereafter bolls form from theseabnormal flowers and develop normally. At this time, about 78% of thesebolls contain hybrid seed, as evidenced by the production of red leafseedlings in a subsequent germination test.

(b) The procedure is followed with the variety of cotton known as Popeusing the same 1% solution. The interplantings are of the red leafvariety. About 28 days after spraying 87% of the bolls develop fromcrosspollinated flowers, owing to the absence of normal pollen followingthe chemical treatment. Again, this percentage is validated bysubsequent seed germination tests.

This application is a continuation in part of application Serial No.658,253, filed May 10, 1957.

We claim:

1. The process of producing hybrid cotton seed which comprises plantingat least two varieties of cotton in close proximity to each other butsufiiciently segregated to permit application of a chemical to onevariety without substantial contact of the chemical with the othervariety, applying to one of the said varieties before opening of flowersand buds thereof an aqueous solution of a compound which forms in wateran anion from the class consisting of CCl =CClCOO-- and CH CICHCICOO-said solution containing from 0.1% to 5% of said compound and beingapplied at a rate below that which causes serious injury to the plantunder the conditions existing at the time of application, continuinggrowth of the two varieties of plants with development of flowersthereon, maturing and separately collecting the seed of the treatedplants when the seed has matured.

2. The process of producing hybrid cotton seed which comprises plantingat least two varieties of cotton in close proximity to each other butsufiiciently segregated to permit application of a chemical to onevariety with out substantial contact of the chemical with the othervariety, applying to one of the said varieties before opening of flowersand buds thereof an aqueous solution of a compound which forms in waterthe anion CCl =CClCOO" said solution containing from 0.1% to 5% of saidcompound and being applied at a rate below that which causes seriousinjury to the plant under the conditions existing at the time ofapplication, continuing growth of the two varieties of plants withdevelopment of flowers thereon, maturing and separately collecting theseed of the treated plants when the seed has matured.

3. The process of producing hybrid cotton seed which comprises plantingat least two varieties of cotton in close proximity to each other butsufficiently segregated to permit application of a chemical to onevariety without substantial contact of the chemical with the othervariety applying to one of the said varieties before opening of flowersand buds thereof an aqueous solution of a compound which forms in waterthe anion CH CICHCICOO- said solution containing from 0.1% to 5% of saidcompound and being applied at a rate below that which causes seriousinjury to the plant under the conditions existing at the time ofapplication, continuing growth of the two varieties of plants withdevelopment of flowers thereon, maturing flowers and seeds thereon, andseparately collecting the seed of the treated plants when the seed hasmatured.

4. The process of producing hybrid cotton seed which comprises plantingat least two varieties of cotton in close proximity but sufficientlysegregated to permit application of a chemical to one variety withoutsubstantial contact of the chemical with another variety, applying toone of said varieties before the opening of flower buds thereon acomposition containing a chlorinated aliphatic compound which forms inwater anions of the formula CCl =CClCOO-, said chlorinated solutionbeing applied at a rate between 1 and 16 pounds per acre, the rate andthe concentration of said compound in said composition being below therate and concentrations which cause serious injury to the treated plantsunder conditions existing at the time of application, allowing theplants to form flowers, fertilizing flowers of said treated plants withpollen from an untreated variety of cotton plant, allowing seed tomature on treated plants, and separately collecting the seed therefrom.

5. A process according to claim 4 in which the compound is sodiumtrichloroacrylate.

6. The process of producing hybrid cotton seed which comprises plantingat least two varieties of cotton in close proximity but suflicientlysegregated to permit application of a chemical to one variety withoutsubstantial contact of the chemical with another variety, applying toone of said varieties before the opening of flower buds thereon acomposition containing a chlorinated aliphatic compound which forms inwater anions of the formula CH ClCHClCOO-, said chlorinated solutionbeing ap plied at a rate between 1 and 16 pounds per acre, the rate andthe concentration of said compound in said composition being below therate and concentrations which cause serious injury to the treated plantsunder conditions existing at the time of application, allowing theplants to form flowers, fertilizing flowers of said treated plants withpollen from an untreated variety of cotton plant, allowing seed tomature on treated plants, and separately collecting the seed therefrom.

7. A process according to claim 6 in which the compound is sodium2,3-dichloropr0pionate.

No references cited. i

1. THE PROCESS OF PRODUCING HYBRID COTTON SEED WHICH COMPRISES PLANTINGAT LEAST TWO VARIETIES OF COTTON IN CLOSE PROXIMITY TO EACH OTHER BUTSUFFICIENTLY SEGREGATED TO PERMIT APPLICATION OF A CHEMICAL TO ONEVARIETY WITHOUT SUBSTANTIAL CONTACT OF THE CHEMICAL WITH THE OTHERVARIETY, APPLYING TO ONE OF THE SAID VARIETIES BEFORE OPENING OF FLOWERSAND BUDS THEREOF AN AQUEOUS SOLUTION OF A COMPOUND WHICH FORMS IN WATERAN ANION FROM THE CLASS CONSISTING OF CCI2=CCICOO-AND CH2CICHCICOOSAIDSOLUTION CONTAINING FROM 0.1% TO 5% OF SAID COMPOUND AND BEING APPLIEDAT A RATE BELOW THAT WHICH CAUSES SERIOUS INJURY TO THE PLANT UNDER THECONDITIONS EXIXTING AT THE TIME OF APPLICATION, CONTINUING GROWTH OF THETWO VARIETIES OF PLANTS WITH DEVELOPMENT OF FLOWERS THEREON, MATURINGAND SEPARATELY COLLECTING THE SEED OF THE TREATED PLANTS WHEN THE SEEDHAS MATURED.